Lipids are molecules that are fundamental to the existence of all living organisms. Lipids are non-polar molecules that are water-insoluble. As such, the term lipids includes a large number of structurally distinct biomolecules, including phospholipids, glycolipids, and sterols, like cholesterol.
Lipids have a variety of biological roles. First, lipids are the major component of biological membranes. Like exterior walls of houses, biological membranes are structurally organized barriers which define and separate cells from the environment and other cells. Like interior walls of houses, biological membranes are structurally organized barriers that compartmentalize and organize the cell's intracellular components.
Biological membranes, however, are not impervious walls. Instead, they are highly selective permeable barriers which regulate the quality and quantity of molecules which are allowed to pass through the membrane. The cell membrane, for example, tightly regulates the amount of water, ions and sugar which can pass into the cell.
One class of lipids which is abundant in all biological membranes is phosphoglycerides. Phosphoglycerides are comprised of a glycerol (a three-carbon alcohol) backbone, two fatty acid chains (long hydrocarbon molecules), and a phosphate. The simplest phosphoglyceride that can be formed is phosphatidic acid. Phosphatidic acid has two fatty acid chains esterified to the hydroxyl groups at the C-1 and C-2 positions of glycerol, respectively. The C-3 hydroxyl group of glycerol is esterified to phosphoric acid. While phosphatidic acid is not a major component of biological membranes, it is a key intermediate in the formation of structurally related phosphoglycerides such as phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, which has a sugar moiety attached to the phosphate.
Second, fatty acid-containing lipids are an energy source for cells and organisms. Fatty acids in a series of biological reactions are oxidized by certain cells to yield large amounts of energy necessary to carry out essential biological functions. Fatty acids used for fuel are stored as triacylglycerols (which are sometimes referred to as "triglycerides"), or neutral fats. Like phosphatidic acids, triglycerides have a glycerol backbone. Rather than having two fatty acid and a phosphate group, however, triglycerides contain three fatty acid chains.
Triglycerides are an efficient way to store large quantities of energy, and thus are the major energy reservoir in humans and other mammals. The complete oxidation of a typical fatty acid yields approximately 9 kcal/g, as compared to about 4 kcal/g for proteins and carbohydrates. Moreover, unlike carbohydrate energy stores, triglycerides are anhydrous (i.e., do not contain water). Consequently, a gram of triglycerides contains more than six times the energy of one gram of carbohydrate. Taken together, triglycerides account for about 80% of all the energy of an average individual.
Finally, lipids participate in cell-cell communication, differentiation and proliferation. Normal development and function in living organisms requires interactions between cells and the molecules in the surrounding environment. One way cells communicate is via molecules, called transmembrane proteins, that span the cell's biological membrane. When the portion of the transmembrane protein which is outside of the cell encounters specific molecules in the surrounding environment, it undergoes conformational changes that trigger a biological cascade inside the cell.
The binding or interaction of a molecule in the environment with a transmembrane protein frequently activates a membrane-bound enzyme called phospholipase C. The activation of phospholipase C is at the center of many major biological events. For example, the activation of phospholipase C is correlated with cell proliferation. Vasopressin, prostaglandin F2, and bombesin, which stimulate cell proliferation, stimulate phospholipase C. In addition, phospholipase C plays a role in activation of T lymphocytes of the immune system and fertilization of eggs.
Phospholipase C exerts its biological effects by catalyzing a reaction which cleaves the sugar moiety of the cell membrane lipid phosphatidylinositol 4,5 bisphosphate. The reaction releases diacylglycerol (DAG) and inositol triphosphate. Diacylglycerol and inositol triphosphate, referred to as second messengers, in turn, activate other molecules within the cell. Diacylglycerol, for example, activates an enzyme called protein kinase C (PKC), which is central to numerous biological processes, including the regulation of cell growth and differentiation.
DAG is at the heart of lipid-mediated biological events. See U.S. Patent application Ser. No. 08/841,483 filed Apr. 22, 1997. Diacylglycerol is a precursor to phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, which are indispensable components of biological membranes. In addition, diacylglycerol is a precursor to triglyceride biosynthesis and, therefore, is central to energy stores of organisms. Finally, diacylglycerol is a second messenger which binds and activates protein kinase C, leading to numerous biological events.
The proper regulation of diacylglycerol in cells, therefore, is critical for proper biological function. Abnormally high or low levels of diacylglycerol would be predicted to alter the lipid biosynthesis and the activity of enzymes that depend on diacylglycerol, like PKC.
Diacylglycerol kinase (DGK), which catalyzes phosphorylation of DAG to phosphatidic acid (PA), is thought to be a key enzymes in the regulation of DAG levels and, as a result, to be responsible for attenuating the activation of PKC. For example, a constitutively elevated level of DAG (leading to activated PKC) is common in transformed cells, and experimental overexpression of DGK.alpha. decreased the elevated DAG level in ras-transformed fibroblasts. T. Fu et al. (1992), FEBS Lett.307:301-304. This type of experiment suggests that conversion of DAG to PA suppresses a mitogenic signal, but this conclusion is complicated by the fact that PA itself may be mitogenic. W. Moolenaar et al. (1986), Nature 323:171-173. PA has been implicated in the regulation of DNA synthesis, in the induction of c-myc, c-fos, and platelet-derived growth factor; in cAMP formation; and in modulating the activity of n-chimaerin and NF1. Thus, since both DAG and PA can act as second messengers, their interconversion is likely to be tightly regulated.
DGK activities have been detected from a variety of tissues and organisms from Arabidopsis thaliana and E. coli to mammals. Eight mammalian DGKs have been identified and characterized; they differ in their activators, expression patterns, substrate specificity and structural domains. DGKs can be divided into five subfamilies according to distinctive structural motifs. Type I includes DGK.alpha., .beta., and .gamma., which have E-F hand motifs at their N-termini and are stimulated by Ca.sup.++, although the binding affinity for Ca.sup.+ differs among these three isoforms. DGK.delta. and .eta. are type II DGKs; each has a pleckstrin homology domain ("PH domain") at its N-terminus instead of an E-F hand motif. PH domains have been found in a number of proteins involved in signal transduction and serve as sites of protein-protein and protein-phospholipid interactions. The third type of DGK, DGK.epsilon., has the simplest structure in the DGK family and shows substrate selectivity for DAG with an arachidonoyl residue at the sn-2 position. Type IV is typified by DGK.zeta., which has four ankyrin repeats at its C-terminus and a region similar to the MARCKS phosphorylation site domain. The Drosophila DGK2, rdgA gene also belongs to this group, and it is expressed almost exclusively in the retina. I. Masai et al. (1993), Proc. Natl. Acad. Sci. USA. 90:11157-11161 ("Masai et al."). A mutation in rdgA causes degeneration of photoreceptor cells and blindness. Id. Interestingly, the photoreceptor cells degenerate regardless of whether the cells are exposed to light or not, implying that Drosophila DGK2, rdgA may be required for more than just a light-signaling cascade, and a recent study showed that the degeneration begins with disruption of organelles responsible for the transportation of phospholipids to the photoreceptor membrane. I. Masai et al. (1997), J. Neurobiol. 32:695-706. One of the mutations that results in the retinal degeneration phenotype in Drosophila is a stop codon before the ankyrin repeats, and these motifs are known to mediate protein recognition. V. Bennett (1992), J. Biol. Chem. 268:1501-1504. Proteins containing ankyrin repeats are involved in a variety of cellular processes such as gene regulation and cell cycle control. The type V DGK, DGK.theta., contains three cysteine-rich repeats instead of the typical two, and a ras-binding domain.
From the foregoing, it will be appreciated that it would be an advancement in the art to identify and characterize nucleic acid sequences that code for enzymes that catalyze the conversion of DAG to PA. It would be a further advancement to identify nucleic acid sequences coding for such enzymes that regulate signal transduction in specific tissues, such as retina and brain. It would be a further advancement in the art to provide methods of detecting DGK mRNA and proteins in a cell. Finally, it would also be an advantage to provide means for regulating cell proliferation by decreasing the pools of diacylglycerol available to activate PKC.
Such nucleic acid sequences and methods are disclosed and claimed herein.